The basic premise of this study is that the vascular endothelium represents an important, yet understudied and therapeutically untapped, subset of peripheral, non-hematopoietic 'semi-professional' antigen presenting cells (APCs). Similar to hematopoietic 'professional' APCs (but distinct from most other cell types) endothelial cells constitutively express essential peptide (MHC-I, MHC-II) and lipid (CD1d) antigen presentation molecules, along with critical co-stimulators and co-inhibitors. However, they lack the co-stimulators (CD80, CD86) essential for initiating adaptive immune responses (i.e., for priming of naive T cells). Thus endothelial APC functions would seem to be geared for later effector/memory/tolerogenic stages of responses within the periphery. Indeed, a range of in vitro and in vivo studies have supported this hypothesis and implicated endothelial APC function in autoimmune/inflammatory/cardiovascular pathologies such as atherosclerosis, arthritis, lupus, multiple sclerosis, diabetes, Crohn's disease, allograft rejection and myocarditis. However, overall contributions of endothelial cells as APCs has remained poorly appreciated, controversial and in many cases completely ignored. This is striking given their ubiquitous tissue distribution, constitutive interaction with circulating lymphocytes and that they, in fact, outnumber professional hematopoietic APCs (conservatively) by ~1000-fold. The central objective of this study is to build the first systematic framework for understanding roles for endothelial APCs in adaptive immune responses and inflammatory pathology, with the ultimate goal of harnessing this knowledge to improve on emerging and exciting APC-dependent immune-modulatory therapeutic strategies. Our preliminary studies have established in vitro model systems that confirm the ability of endothelial cells to activate CD4+ and CD8+ effector/memory cells and that demonstrate for the first time that endothelial cells can effectivel present lipid antigen to, and activate NKT cells. Additionally, we have defined novel endothelial cell-lymphocyte immunological synapse structures that mediate these interactions. Furthermore, we have generated novel endothelial-specific MHC-II and CD1d murine knockout strains, and with the former, generated the first evidence of an unambiguous role for endothelium in adaptive immunity in vivo. This proposal will extend these preliminary studies, focusing on the potent and interrelated CD4+ T helper and NKT cytokine-driven antigen responses. A series of in vitro studies will delineate basic roles of endothelial APCs, their heterogeneous function in distinct vascular beds, their plasticity in response to critical pathologic stimuli and their sensitivity to pharmacologic-/antibody-based intervention. Finally, murine transplant and infection models will be employed, along with our endothelial MHC-II and CD1d knockout strains, to i) generate expanded proof-of-principle demonstration of endothelial APC-specific functions in adaptive immunity and ii) to evaluate the critical translational potential of endothelial cells in mediatingin vivo immunomodulatory effects of emerging therapeutics. These studies will be highly significant in that they will open fundamental new avenues for understanding and for pharmacologically addressing broad ranging inflammatory/cardiovascular diseases.